Our research group has assessed the global expression patterns of miRNAs in both tumor and normal samples from patients with head and neck squamous cell carcinoma (HNSCC); we identified miR-375 as the most consistently down-regulated miRNA in tumor samples when compared to paired normal samples. Patients in the lowest quartile of miR-375 expression had significantly decreased disease-specific survival, increased frequency of local regional recurrence and distant metastasis. I hypothesize that miR-375 suppresses the invasive phenotype of HNSCC through repression of the levels of specific protein targets. The highly significant association of lower miR-375 expression in patients with poor prognosis and distant metastasis led me to want to investigate how miR-375 expression causes alterations in the invasive properties of HNSCC. I have observed that precursor miR-375 transductants have reduced invasion in vitro. Furthermore, preliminary gene expression microarray analysis of the transductants has identified the down-regulated expression of genes having functions associated with cell motility and invasion; the expression of some of these genes also have been previously been correlated with HNSCC patient survival. This proposal addresses the invasive phenotype hypothesis with the following aims: 1) Utilize stable lentiviral transductants to investigate the effects of miR-375 on the invasive properties of HNSCC cell lines. Stable transductant cell lines will be used to assess whether miR-375 expression levels affects growth, invasion, chemotaxis and matrix degradation in vitro, as well as, three-dimensional embedded growth, three-dimensional invasion with inverted invasion and organotypic invasion assays. In addition HNSCC floor-of-mouth xenografts generated from UMSCC47 transductant cell lines will be used to evaluate the consequence of miR-375 expression levels on tumor growth, in vivo invasion and metastasis. 2) Identify specific miR-375 target genes and altered signaling pathways in HNSCC. Stable lentiviral transductants will be used to identify potential target genes of miR-375 by analyzing both RNA expression with whole genome microarrays and translationally repressed putative targets of miR-375 by SILAC (stable isotope labeling of cells in cell culture). Ingenuity Pathway Analysis will be used to identify cellular functions and signaling pathways that the potential targets of miR-375 are involved. The expression levels of the putative targets will be confirmed by qRT-PCR and/or western blot analysis. The interaction between miR-375 and the putative target mRNA transcripts will be confirmed by luciferase reporter assays. Our group has generated a database of mRNA microarray data and global proteomic data on primary tumors from patients along with clinical data on each sample. The disease relevance to any candidate miR-375 target identified in my analysis will be assessed by correlations of cell line data with patient-derived data from primary tumors. Functional validation of the phenotypic effects of the down-regulation of putative targets of miR-375 will be performed by siRNA knockdown and rescue of the targets in the transductant cells.